Sputum analysis method

ABSTRACT

An in vitro diagnostic technique for quantitative analysis of human sputum, such as lung fluid, for the presence of microbial pathogens is provided wherein a sputum sample is contacted with a minor quantity of nontoxic saponin to degrade the viscosity of the sputum and thereafter the sputum is thoroughly admixed to distribute microbial pathogens generally uniformly therein. The sputum can be diluted as desired and then subjected to conventional analytical techniques, i.e., plating on growth media and recording results.

BACKGROUND OF THE INVENTION

This invention relates to the quantitative analysis of human sputums for the presence of microbial pathogens. In another aspect, this invention relates to a novel technique of liquefying sputum specimens with a saponin mucolytic agent.

The condition of pneumonia in a human patient generally comprises an acute inflammatory condition of a lung or lungs which can be caused by microbial pathogens such as bacteria or viruses as well as chemical irritants or foreign bodies. In order to determine the causal agent in a patient with a presumptive diagnosis of pneumonia, samples of lung fluid are required. The ideal specimens of lung fluid are homogeneous samples which can be obtained by surgical intervention, for example, transtrachael aspiration. As is typical, surgical intervention techniques are time consuming and present a risk to the patient. Fatal reactions have been reported resulting from transtrachael aspiration. For example, hypotoxic patients and those suffering from debilitating diseases such as blood dyscrasia are especially prone to serious complications from transtrachael aspiration.

As a consequence, most physicians rely on the early morning couph sputum specimen as the means of obtaining a lung fluid specimen. This technique is simple and very common. Unfortunately, the cough sputum specimen can be readily contaminated by the normal flora in the mouth, nose, posterior pharynx, and stomach. Furthermore, sputums from patients with pneumonia are very viscous and heterogeneous in nature and therefore difficult to disperse in a reproducible manner. Quantitative analysis of sputum cannot be accomplished on the very viscous heterogeneous sputums because it is generally necessary in quantitative analysis of specimens like sputum to dilute the specimen and thereafter plate minor portions of the specimen on various growth media and then determine the type and number of colonies of microbial organisms which result on the media. Generally, the maximum number of colonies which can be effectively counted on a petri dish is about 300. Furthermore, it has been accepted that normal sputum samples can contain contaminating microorganisms in quantites up to 10⁵ per milliliter, whereas, as causative organisms of pneumonia microorganisms are generally present in quantities greater than about 10⁶ per milliliter. Therefore, before a sputum specimen can be quantitatively analyzed, it must be diluted in a manner such that the microbial pathogens are uniformly dispersed in the resulting diluted sample. Accordingly, if a minor portion of this diluted sputum specimen is quantitatively analyzed for the type and number of microbial pathogens, the accurate number of microbial pathogens per milliliter of the sputum sample can be accurately calculated.

Thus, in order to reduce the problem of external contamination and heterogeneity of the sputum sample and provide a quantitative sputum analysis, several attempts have been made to digest the sputum specimen with enzymatic and chemical digestants. Several such digestants have been theretofore tried and all of the digestants have one or more of the following disadvantages: expensive; short shelf life; temperature sensitive; toxic to some or many pathogenic organisms; and require long digestion time. For example, a number of proteolytic enzymes have been tested in both purulent and mucoid sputum. Of such enzymatic materials, trypsin, elastase, and chymotrypsin appear the most effective, and enzymes such as bromelain, ficin or papain were only effective at extreme high concentrations, while plasmin has no detectable effect on sputum viscosity. All such proteolytic enzymes appear to be more effective with mucoid sputums than with purulent sputums. The most widely used digestants for quantitative sputum analysis are aqueous solutions of N-acetylcysteine and Cleland's reagent (1,4-dithio-mesoerithritol). In general, Cleland's reagent exhibits greater mucolytic activity than N-acetylcysteine at lower concentrations, but Cleland's reagent generally loses its mucolytic activity in relatively short periods of time in aqueous solution. Furthermore, both of these reagents are somewhat toxic to microbial pathogens at concentrations needed for rapid digestion of sputum.

Consequently most sputum specimens are processed by a nonquantitative streaking technique, which generally involves streaking the heterogeneous sputum specimen on various growth media. These techniques lead to a substantial number of false positive cultures, and in many instances, overgrowth of the pathogenic organisms by contaminating microorganisms.

SHORT STATEMENT OF THE INVENTION

According to the invention, I have discovered that certain purified saponins exhibit mucolytic activity and will effectively degrade sputum samples including both purulent and mucoid sputums and uniformly disperse microbial pathogens therewithin without harming the pathogens.

According to one embodiment of the subject invention, a novel technique for the quantitative analysis of sputums is provided which includes the steps of contacting the sputum specimen after it is collected with an effective mucolytic portion of a nontoxic saponin to convert the sputum specimen to a substantially uniform viscosity and mixing the specimen such that any pathogens are uniformly distributed therewithin and thereafter conducting quantitative analysis techniques on the resulting specimen.

DETAILED DESCRIPTION OF THE INVENTION

The nontoxic saponin which is utilizable within the scope of the subject invention can be purified by the method disclosed in U.S. Pat. No. 3,883,425 which patent is herein incorporated by reference into this specification.

Saponins are glycosides widely distributed in plants and are capable of forming oil-in-water emulsions, and act as protective colloids. Each saponin molecule consists of a sapogenin which constitutes the aglucone moiety of the molecule, and a sugar.

The sapogenin is either a triterpenoid (usually a pentacyclic structure, such as quillaic acid), or a stearoid structure (usually having a spiro acetal side chain as in diosgenin). The sugar portion of the saponin glucoside includes one or more sugars such as glucose, sucrose, xylose, a pentose or methyl pentose, or other sugars. On hydrolysis, the saponins yield the sapogenin and one or more of these sugars.

Commercially available saponins comprise a white to brown amorphous powder which is pungent, and has a disagreeable taste and odor. This powder is very soluble in water and foams very strongly when shaken with water.

Commercial saponins are prepared by extracting plant tissue with water and/or other organic solvents, such as alcohol and recovering saponin by precipitation, recrystallization and the like. Saponins are widely distributed in plants. For example, saponins are very widely distributed in the plant family caryophyllaceae. Specific examples of saponin sources include soap bark, panama wood, soap berry, liquorice, and the like. A specific example of a process for producing commercial saponin is described in Kingzette's Chemical Encyclopedia, D. H. Hey, 9th Edition, (1966) which includes either extracting powdered soap bark (Quillaria saponaria) with hot alcohol or by boiling powdered dry aqueous extract of such bark with alcohol, and allowing saponin to crystallize from the alcohol upon cooling.

Saponins are practically nontoxic to humans upon oral ingestion, but act as powerful hemolytics when injected into the bloodstream, thereby dissolving the red blood corpuscles. Because of this characteristic, saponins have been conventionally used in hemotology laboratories for lysing the red cells whenever their presence interferred with other procedures, e.g., hemoglobin determinations and white cell counts.

As set forth in said patent, U.S. Pat. No. 3,883,425, a technique is provided for removing microbial toxins from saponin which is extracted from a plant source by removing consitutents from an aqueous solution of said saponin which have an apparent molecular weight of less than about 600, e.g., a molecular size in aqueous solution between about 140 to about 600. One technique disclosed in said patent is to form an aqueous solution of a commercial saponin extract from plants and thereafter pass the solution through a microporous filter membrane which has a mean actual pore size no smaller than about 11 angstroms in diameter and no larger than about 24 angstroms in diameter. The aqueous solution which passes through the microporous filter membrane will contain the antimicrobial toxin, and the filter will exclude the saponin material.

I have now found that such purified saponin plant extracts are effective mucolytic agents in that they will efficiently degrade all types of sputum and disperse microbial pathogens therewithin without harming the pathogens.

In carrying out the method of the subject invention, the sputum sample is collected by any conventional technique. Thereafter the sputum is admixed with a minor but effective mucolytic amount of the nontoxic saponin described above. Preferably the saponin is in aqueous solution when admixed with the sputum. The aqueous solution can be of any convenient concentration such that when the desired amount of solution is admixed with the sputum specimen, sufficient saponin will be present to degrade the same. Generally, it is necessary from about 0.1% by weight to about 20% by weight and preferably from about 1.0% by weight to about 10% by weight and even more preferably from about 5% by weight to about 10% by weight of saponin be contained in the resultant mixture of saponin and sputum.

In addition, it is preferable to add to the resultant mixture of saponin and sputum from about 0.1% by weight to about 20% by weight and more preferably from about 5% by weight to about 10% by weight of an antifoamant which is nondeleterious to microbial pathogens. Suitable such antifoam agents include materials sold under the trademarks of Dow X Antifoam B and Dow X H 10. These agents contain chemical polymers of dimethylsiloxane and a sterilizable, e.g., autoclavable, nonionic emulsifier such as the type sold under the trademark of Triton X. The material sold under the trademark of Dow X H 10 may be preferred since it is very stable to sterilization by autoclaving. The saponin effectively degrades all types of sputum both mucoid and purulent very rapidly to form a mixture of uniform viscosity.

The sputum sample is then thoroughly admixed to uniformly disperse the microorganisms therewithin. If desired, portions of the resulting degraded sputum sample can be plated directly on various media to obtain an improved qualitative sputum analysis which is not subject to the substantial number of false-positive cultures and overgrowth of pathogenic flora by contaminating microorganisms as is the conventional qualitative technique which does not utilize degraded sputum.

When conducting an improved quantitative analysis of sputum in accordance with the subject invention, the saponin treated sputum is diluted as desired, e.g., 1:100-1:1000 with phosphate buffered normal saline, pH 7.2 or any other suitable diluent which will not interfere in the analysis technique and is nontoxic to the microbial pathogens. The resulting diluted specimen is thoroughly admixed to assure uniform distribution of the microbial pathogens throughout the solution. Next, samples of the diluted solution are added to conventional growth media and allowed to incubate. Next, any resulting colonies on the media can be identified and counted and by knowing the extent of dilution of the sputum specimen, the total quantity of different types of microbial pathogens can thereby be easily calculated. Alternately, if desired, the nondiluted specimen can be subjected to microscopic examination.

The following examples are presented to more specifically illustrate the subject invention.

EXAMPLE I

In this Example, the toxicity of various known mucolytic agents was compared to that of saponin. In each instance, the strains of bacteria shown in Table 1 below were grown overnight in the growth media indicated in Table 1 and the turbidity of the resultant growth media was adjusted with sterile saline to a barium sulfate standard to approximately 10⁸ bacteria per milliliter. Thereafter the resulting broth was diluted 1:200 in nutrient broth. Next, a 40 weight percent solution of saponin which was purified in accordance with the procedure set forth in U.S. Pat. No. 3,883,425 (to thereby remove toxic contaminants therefrom which have an apparent molecular weight of less than about 600 in aqueous solution) was added to the type of growth broth for the particular bacteria as indicated in Table 1. Thereafter, 1 milliliter of each bacterial-growth broth suspension was added to 1 milliliter of saponin solution described above to give a final concentration of 20% by weight saponin, and then was incubated overnight at 37° C. A control and test suspension was also streaked on rich agar containing 1 weight percent glucose, or blood agar.

The other compounds tested for toxicity were Cleland's reagent (1,4-dithio-mesoerithritol), N-acetylcysteine, and sodium lauryl sulfate. These mucolytic agents were prepared in the concentrations and in the growth broth for each of the microorganisms as indicated in Table 1 and tested for toxicity in the same manner as the saponin described above.

If the particular bacteria grew in the broth containing the indicated quantity of mucolytic agent, the run was repeated to see if growth could be duplicated in a second run. If growth was not obtained either the first or second time, a third run was made in an attempt to grow the bacteria. In some instances, a third run was made even though bacteria grew in the prior two runs. Therefore, in the table a plus indicates a successful growth run and a minus indicates a negative growth run. More specifically:

+++ = grew three out of three times

--- = did not grow once out of three times

--+ = grew one out of three times

-++ = grew twice out of three times

The results are set forth in Table 1 below:

                                      Table 1                                      __________________________________________________________________________                              Mucolytic Agent and Percent Concentration                             Type of  Saponin                                                                              Cleland's N-acetylcysteine                                                                         Sodium lauryl sulfate       Organism        Growth Broth*                                                                           20%   0.1% 0.5% 0.5% 5.0% 2.5%  10.0%                 __________________________________________________________________________     Escherichia coli I                                                                             1        ++    +++  -++  +-+  ---  +++   +++                   Staphylococcus XIII                                                                            1        ++    +++  ---  ++-  ---  ---   ---                   Pseudomonas species                                                                            1        ++    +++  ---  ++-  ---  +-+   +-+                   Candida species 1        ++    +++  -++  +++  ---  +--   ---                   Torulopsis glabrata                                                                            1        ++    +++  -+-  +++  ---  ---   ---                   Streptococcus pyogenes                                                                         2        ++    ++   --   +-   +-   --    --                    Streptococcus pneumoniae                                                                       3        ++    ++   --   --   --   --    --                    Citrobacter freundii                                                                           1        ++    +++  +++  -++  ---  +++   +++                   Klebsiella pneumoniae                                                                          1        ++    +++  -++  +++  ---  +++   +++                   Enterobacter aerogenes                                                                         1        ++    +++  -++  +++  ---  +++   +++                   Salmonella cholerasuis                                                                         1        ++    +++  -++  --+  ---  +++   +++                   Proteus mirabilis                                                                              1        ++    +++  -++  +++  ---  +++   +++                   Listeria monocytogenes                                                                         4        ++    +++  ++-  +--  ---  ---   ---                   Neisseria meningitidis                                                                         4        ++    -    -    -    -    -     -                     Brucella suis   4        ++    -+   --   +-   --   --    --                    Bacteroides fragilis                                                                           5        ++    ++   +-   +-   --   --    --                    Clostridium perfringins                                                                        5        ++    ++   +-   +-   -    -     -                     Propionibacterium shermanii                                                                    5        ++    +-   +-   --   - -  --    --                    Mycoplasma hominis                                                                             6        ++    NT   NT   NT   NT   NT    NT                    __________________________________________________________________________       *1 grew in rich broth containing 1 wt % glucose.                               2 grew in Todd - Hewett broth and thereafter streaked on blood.               3 grew in rich broth containing 1 wt % glucose and thereafter streaked on      blood.                                                                         4 grew in rich broth containing 1 wt % glucose and thereafter streaked on      blood in a CO.sub.2 jar.                                                       5 grew in a peptone yeast glucose maltose broth and thereafter streaked o      blood in an anaerobic jar.                                                     6 originally grew in a PPLO broth and thereafter streaked on PPLO agar in      a CO.sub.2 jar.                                                                NT not tested.                                                           

As can be seen from Table 1 above, the relatively high concentration of the purified saponin did not kill or inhibit the growth of any of the micoorganisms tested. However, each of the other mucolytic agents tested prevented growth of several of the microorganisms at much lower concentration than the saponin. It should also be noted that even at recommended use concentration of presently used mucolytic agents (0.5% N-acetylcysteine and 0.1% Cleland's reagent) toxicity against several organisms was noted.

EXAMPLE II

Clinical analyses of several sputum samples was conducted after the samples had been dissolved in nontoxic saponin and the results compared to the routine sputum analysis that is conventionally conducted in a typical hospital. Initially, an early morning sputum specimen was collected and a wet prep and smear were made to grade the sputum and to observe characteristics thereof such as spores. The sputums were graded 1 through 4 according to the following protocol:

Grade 1: saliva -frothy, clear, colorless, mostly epithelial cells on a 40X field.

Grade 2: mucoid -colorless, clear to translucent, majority of eosinophils and macrophages, polymorphonuclear cells less than 60%, and less than 1/2 of a 40X field having cells.

Grade 3: mucopurulent - colorless to greenish, slightly translucent, and approximately 1/2 of the field filled with cells.

Grade 4: purulent - yellow or greenish, cloudy, polymorphonuclear cells more than 70%, and greater than 1/2 of the field covered with cells.

In each instance, the cellular density was determined by observing what percentage of a 40X field is filled with cells on a wet mount sample. The cytology is determined by staining (Papanicolaou) a slide, counting 200 cells of the sample and identifying the polymorphonuclear cells (PMN), eosinophils, basophils, macrophages, monocytes, and lymphocytes.

After each sputum sample was graded, a routine analysis was run thereof which includes making 6 semi quantitative streaks with part of the sputum sample on a blood agar plate (BA); on a chocolate agar plate (CHOC); on a eosin methylene blue plate (EMB); on a mannitol salts plate (MAN); on a mannitol salts containing penicillin plate (MAN-PEN); and on a Sabourand's + gentamicin plate (SDA-GENT), and allowing any bacteria to grow on the plate and thereafter identifying the same. The remaining part of each specimen was thereafter digested with an equal amount of 10 weight percent saponin (which has been purified in accordance with U.S. Pat. No. 3,833,425) and 0.1 m sodium citrate, and an antifoaming agent (Antifoam B Emulsion - Dow Corning) to give a final concentration of 5% by weight saponin therein. Thereafter the saponin-sputum mixture is vortexed for at least 30 seconds.

Next, the original digested sputum was streaked onto the following plates: BA; CHOC; EMB; MAN; MAN-PEN; and SDA-GENT. Thereafter, two 1:100 dilutions were made from a portion of the digested sputum into saline bottles to give 10² and 10⁴ dilutions, e.g., 0.1 milliliters sputum was placed into a 9.9 milliliters saline for the 10² dilution, and 0.1 milliliters from the 10² dilution was placed into 9.9 milliliters of saline to make the 10⁴ dilution. Next, a 0.01 milliliter loop was used to streak from the 10⁴ dilution bottle on to the following plates: BA; CHOC; EMB; MAN; MAN+PEN; and SDA+GENT, to give a final definite dilution of 10⁶. Thereafter, another 0.01 milliliter sample was streaked onto SDA gentamicin from the 10² dilution bottle to give a definite dilution of 10⁴. All the plates were incubated and read in 24 and 48 hours. The SDA plates were also read after one week. The plates were interpreted as follows:

a. an interpretation of normal flora (normal level and type of microorganisms found in nasopharyngeal passages) for Alpha streptococcus and Neissera and diphtheroids occurring in the 10⁶ and 10⁷ range.

b. positive infection for all other nonyeast organisms (other than set forth in (a) above) occurring in the 10⁶ and greater range.

c. 10⁵ was interpreted as 1-5 organisms found randomly on a set of plates as a possible positive.

10³ and upon which is 1 to 5 organisms found randomly on a set of plates is a possible positive for Candida Albicans, and a positive for all other yeasts.

The results are set forth in Table 2 below.

                                      Table 2                                      __________________________________________________________________________     CLINICAL RESULTS OF SPUTUM ANALYSIS BY                                         ROUTINE AND QUANTITATIVE ANALYSIS                                              Case                                                                              Grade                               Quantitative                            No.                                                                               Sputum                                                                              Routine Analysis   Conclusion  Analysis    Conclusion                  __________________________________________________________________________     1  2    Light Enterococcus, light Kleb-                                                                   Normal Flora                                                                               Normal Flora                                                                               Normal Flora                        siella pneumoniae, light                                                       Staphylococcus epidermidis,                                                    light Pseudomonas                                                      2  4    Light Staphylococcus aureus                                                                       Normal Flora                                                                               2 × 10.sup.7                                                                         Possible positive                                                  seria perflava                                                                             for Neisseria                                                                  perflava                    3  1    Moderate Serratia marcescens,                                                                     Significant 1 × 10.sup.6                                                                         Positive for                        light Pseudomonas  growth Serra-                                                                              monas aeruginosa,                                                                          Pseudomonas aeru-                                      tia marcescens                                                                             1 × 10.sup.5                                                                         ginosa, possible                                                   marcescens  positive for                                                                   Serratia marcescens         4  2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                5  2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                6  2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                7  1    Light Pseudomonas, light Candida                                                                  Normal Flora                                                                               1 × 10.sup.5                                                                         Possible positive                   albicans                       monas aerugin-                                                                             for Pseudomonas                                                    osa         aeruginosa                  8  4    Heavy Klebsiella pneumonia,                                                                       Positive for                                                                               2.5 × 10.sup.5                                                                       Positive for                        light Candida tropi-                                                                              Klebsiella  (Candida krusei                                                                            yeast, Kleb-                        calis, light Enterococcus                                                                         pneumoniae  and Torulopsis                                                                             siella pneumoniae                                                  glabrata), 1 × 10.sup.8                                                              and Enterococcus                                                   Klebsiella pneu-                                                                           (D)                                                                monia, 4 × 10.sup.7                                                      Enterococcus                            9  4    Moderate Candida albicans                                                                         Positive for                                                                               TNTC Hansenula                                                                             Positive for yeast,                                    Candida albicans                                                                           polymorpha, 1 × 10.sup.5                                                             possible positive                                                  Klebsiella pneu-                                                                           for Klebsiella                                                     moniae      pneumoniae                  10 1    Normal Flora       Normal Flora                                                                               1 × 10.sup.5                                                                         Normal Flora                                                       lococcus aureus                         11 3    Moderate Klebsiella pneumoniae,                                                                   Positive for                                                                               1 × 10.sup.6                                                                         Positive for Candida                light Candida albicans                                                                            Klebsiella  pneumoniae, 1 × 10.sup.5                                                             albicans, possible                                     pneumoniae  Candida albicans                                                                           positive for Kleb-                                                             siella pneumoniae           12 1    Light Pseudomonas fluorescens,                                                                    Normal Flora                                                                               1 × 10.sup.6                                                                         Positive for Candida                light Candida albicans         albicans, 1 × 10.sup.6                                                               albicans and Pseu-                                                 Pseudonomas fluo-                                                                          domonas fluorescens,                                               rescens, 1 × 10.sup.5                                                                possible positive                                                  Klebsiella pneu-                                                                           for Klebsiella pneu-                                               moniae      moniae                      13 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                14 3    Moderate Klebsiella pneumoniae,                                                                   Positive for Kleb-                                                                         1 × 10.sup.6                                                                         Positive for Candi-                 light Candida albicans                                                                            siella pneumoniae                                                                          albicans    da albicans                 15 2    Light Staphylococcus aureus,                                                                      Normal Flora                                                                               1 × 10.sup.6                                                                         Positive for Candida                light Candida albicans         albicans,  1 × 10.sup.6                                                               albicans and Staphy                                               Staphylococcus                                                                             lococcus aureus                                                    aureus                                  16 2    Candida albicans   Positive for Can-                                                                          1 × 10.sup.3                                                                         Possible positive                                      dida albicans                                                                              albicans    for Candida albi-                                                              cans                        17 2    Light Beta streptococcus, light                                                                               2 × 10.sup.5                                                                         Positive for Toru-                  Staphylococcus aureus, light   sis magii, 4 × 10.sup.5                                                               lopsis magii and                   Candida albicans and Candida trop-                                                                            Candida albicans                                                                           Candida albicans                    icalis                                                                 18 4    Haemophilus influenzae                                                                            Positive for                                                                               6 × 10.sup.8                                                                         Positive for Haemo-                                    Haemophilus philus influenzae,                                                                         philus influenzae,                                     influenzae  1 × 10.sup.3                                                                         possible positive                                                  glabrata    for Torulopsis gla-                                                            brata                       19 1    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                20 3    Heavy Enterobacter cloacae, heavy                                                                 Positive for Enter-                                                                        >10.sup.8  Klebsiella                                                                      Positive for Kleb-                  Klebsiella pneumoniae, heavy                                                                      bacter cloacae,                                                                            pneumoniae, >10.sup.8                                                                      siella pneumoniae,                  Staphylococcus epidermidis                                                                        Klebsiella pneu-                                                                           Enterobacter                                                                               Enterobacter cloa-                                     moniae and Staphy-                                                                         cloacae, 10.sup.5                                                                          cae, Candida krusei,                                   lococcus epidermi-                                                                         dida krusei, 10.sup.5                                                                       Torulopsis glabrata                                   dis         Torulopsis glab-                                                                           and Candida tropi-                                                 rata, 10.sup.4 Candida                                                                     calis                                                              tropicalis                              21 3    Normal Flora       Normal Flora                                                                               1 Penicillium                                                                              Normal Flora                                                       humulii                                 22 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                23 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                24 2    Light Candida tropicalis                                                                          Normal Flora                                                                               2.5 × 10.sup.4                                                                       Positive for Candi-                                                tropicalis, 1.5 ×                                                                    da tropicalis and                                                  10.sup.4 Torulopsis                                                                        Torulopsis glabrata                                                glabrata                                25 4    Light Klebsiella pneumoniae, 3                                                                    Normal Flora                                                                               ˜ 10.sup.5                                                                           Possible positive                   col. Staphylococcus aureus     pneumoniae  for Klebsiella                                                                 pneumoniae                  26 3    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                27 4    Light Candida tropicalis                                                                          Normal Flora                                                                               10.sup.6 Escherichia                                                                       Possible positive                                                  coli, 10.sup.3 Toru-                                                                       for Escherichia                                                    lopsis glabrata                                                                            coli and Torulop-                                                              sis glabrata                28 4    Normal Flora       Normal Flora                                                                               6 × 10.sup.7                                                                         Possible positive                                                  philus, 1 × 10.sup.3                                                                 for Haemophilus                                                    Torulopsis gla-                                                                            and Torulopsis                                                     brata       glabrata                    29 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                30 3    Moderate Acinetobacter calcoaceti-                                                                Positive for                                                                               2 × 10.sup.8                                                                         Positive for                        cus (H)            Acinetobacter                                                                              bacter calcoa-                                                                             Acinetobacter                                          calcoaceticus                                                                              ceticus (H),                                                                               calcoaceticus                                          (H)         1 × 10.sup.8                                                                         and  Haemophilus                                                   philus                                  31 4    Normal Flora       Normal Flora                                                                               1 × 10.sup.8                                                                         Positive for                                                       coccus      Diplococcus                 32 3    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                33 2    Candida albicans   Positive for Can-                                                                          1 × 10.sup.5                                                                         Positive for Can-                                      dida albicans                                                                              albicans    dida albicans               34 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                35 3    Light Enterobacter cloacae                                                                        Normal Flora                                                                               10.sup.6 Pseudomonas                                                                       Possible positive                                                  aeruginosa, 10.sup.5                                                                       for Pseudomonas                                                    Proteus mirabilis                                                                          aeruginosa and Pro-                                                            teus mirabilis              36 3    Light Proteus vulgaris                                                                            Normal Flora                                                                               1 × 10.sup.6                                                                         Possible positive                                                  vulgaris    for Proteus vul-                                                               garis                       37 3    Light Candida albicans                                                                            Normal Flora                                                                               Normal Flora                                                                               Normal Flora                38 2    Normal Flora       Normal Flora                                                                               1 × 10.sup. 3                                                                        Possible positive                                                  albicans    for Candida albi-                                                              cans                        39 3    Moderate Klebsiella pneumoniae,                                                                   Positive for                                                                               10.sup.5 Klebsiella                                                                        Positive for Candi-                 light Enterococcus and Candida                                                                    Klebsiella  pneumoniae, 10.sup.5                                                                       da albicans, possi-                 tropicalis                     Enterococcus 10.sup.5                                                                      ble positive for                                                   Candida albicans                                                                            Enterococcus and                                                              Klebsiella pneu-                                                               moniae                      40 3    Heavy Candida albicans, light                                                                     Positive for                                                                               10.sup.9 aeruginosa, Klebsiella                                                            Positive for Kleb-                  Klebsiella pneumoniae                                                                             Candida albicans                                                                           pneumoniae, >10.sup.5                                                                      siella pneumoniae                                                  Candida albicans                                                                           Candida albicans            41 4                                   10.sup.6 Haemophilus                                                                       Possible positive                                                              for Haemophilus             42 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                43 4    Light Candida albicans                                                                            Normal Flora                                                                               10.sup.5 Candida                                                                           Positive for Candi-                                                cans        da albicans                 144                                                                               4    HeavyCandida albicans                                                                             Positive for Can-                                                                          >10.sup.6 Candida                                                                          Positive for Candi-                                    dida albicans                                                                              cans, 10.sup. 5 Ser-                                                                       da albicans,                                                                   possi--    ratia                                                               liquefa- ble positive                                                          for                                                                ciens, 10.sup.5 Pseudo-                                                                    Serratia liquefa-                                                  monas       ciens and Pseudo-                                                              monas                       45 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                46 4    Proteus mirabilis and gram positive                                                               Positive for Pro-                                                                          10.sup.6 Proteus                                                                           Possible positive                   cocci (unable to isolate)                                                                         teus mirabilis                                                                             mirabilis   for Proteus mira-                                                              bilis                       47 4    Light Klebsiella pneumoniae                                                                       Normal Flora                                                                               10.sup.7 Klebsiella                                                                        Positive for Kleb-                                                 pneumoniae, siella pneumoniae                                                  Basidiomycetes                          48 3    Staphylococcus aureus          Normal Flora                                                                               Normal Flora                49 4    Heavy Klebsiella pneumoniae                                                                       Positive for                                                                               1 × 10.sup.8                                                                         Positive for Kleb-                                     Klebsiella pneu-                                                                           pneumoniae  siella pneumoniae                                      moniae                                              50 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                51 3    Light Klebsiella pneumoniae, light                                                                Positive for Citro-                                                                        1 × 10.sup.6                                                                         Possible positive                   Beta Streptococcus, moderate Citro                                                                bacter freundii                                                                            monas putida                                                                               for Pseudomonas                     bacter freundii                            putida                      52 3    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                53 4    Moderate Candida albicans                                                                         Positive for Candi-                                                                        2 × 10.sup.5                                                                         Positive for Candi                                     da albicans albicans and                                                                               da albicans and                                                    Candida tropicalis                                                                         Candida tropicalis          54 3    Light Candida albicans                                                                            Normal Flora                                                                               1 × 10.sup.3                                                                         Possible positive                                                  albicans    for Candida albi-                                                              cans                        55 2    Heavy Klebsiella pneumoniae, light                                                                Positive for Kleb-                                                                         1 × 10.sup.8                                                                         Possible positive                   Escherichia coli   siella pneumoniae                                                                          species, 1  for Neisseria                                                      Fusarium species                                                                           species                     56 3    Light Klebsiella pneumoniae, light                                                                Normal Flora                                                                               ˜3 × 10.sup.5                                                                  Positive for Candi-                 Candida tropicalis             para-psilosis                                                                              da para-psilosis            57 3    Light Haemophilus, light Candida                                                                  Normal Flora                                                                               1 × 10.sup.3                                                                         Positive for                        albicans                       albicans, 1 × 10.sup.8                                                               Haemophilus possi-                                                 Haemophilus ble positive for                                                               Candida albicans            58 4    Moderate Haemophilus                                                                              Positive for                                                                               1.0 × 10.sup.8                                                                       Positive for                                           Haemophilus philus      Haemophilus                 59 2    Heavy Klebsiella pneumoniae, light                                                                Positive for Kleb-                                                                         1 × 10.sup.6                                                                         Possible positive                   Escherichia coli   siella pneumoniae                                                                          pneumoniae  for Klebsiella                                                                 pneumoniae                  60 4    Light Haemophilus  Normal Flora                                                                               >3 × 10.sup.8                                                                        Positive for                                                       philus      Haemorphilus                61 2    Light Klebsiella pneumoniae                                                                       Normal Flora                                                                               1 × 10.sup.6                                                                         Positive for Kleb-                                                 pneumoniae, 1 × 10.sup.3                                                             siella pneumoniae,                                                 Candida albicans                                                                           possible positive                                                              for Candida albicans        62 4    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                63 4    Haemophilus        Positive for                                                                               1 × 10.sup.7                                                                         Positive for                                           Haemophilus philus      Haemophilus                 64 2    Light Serratia liquefaciens                                                                       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                65 4    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                66 3    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                67 3    Heavy Klebsiella pneumoniae, light                                                                Positive for                                                                               1 × 10.sup.7                                                                         Positive for                        Enterococcus       Klebsiella pneu-                                                                           coccus (D), 3 ×                                                                      Enterococcus (D)                                       moniae      10.sup.8 Klebsiella                                                                        and Klebsiella                                                     pneumoniae  pneumoniae                  68 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                69 2    Moderate Staphylococcus aureus,                                                                   Positive for                                                                               1 × 10.sup.7                                                                         Positive for                        light Alpha streptococcus                                                                         Staphylococcus                                                                             coccus aureus                                                                              Staphylococcus                                         aureus                  aureus                      70 2    Heavy Serratia liquefaciens,                                                                      Positive for                                                                               >10.sup.8 Serratia                                                                         Positive for Ser-                   light Alpha Streptococcus                                                                         Serratia lique-                                                                            liquefaciens                                                                               ratia liquefa-                                         faciens                 ciens                       71 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                72 3    Moderate Enterobacter aerogenes                                                                   Positive for Enter-                                                                        1 × 10.sup.7                                                                         Positive for Enter-                                    obacter aerogenes                                                                          bacter aerogenes,                                                                          obacter aerogenes,                                                 1 × 10.sup.6                                                                         possible positive                                                  liquefaciens                                                                               for Serratia lique-                                                            faciens                     73 2    Normal Flora       Normal Flora                                                                               1 × 10.sup.7                                                                         Positive for Ser-                                                  liquefaciens                                                                               ratia liquefaciens          74 4    Light Klebsiella pneumoniae,                                                                      Normal Flora                                                                               Normal Flora                                                                               Normal Flora                        light Enterococci                                                      75 2    Enterobacter, Candida albicans 1 × 10.sup.4                                                                         Positive for Candi-                                                albicans    da albicans                 76 4    Enterobacter aerogenes, Candida                                                                               >1 × 10.sup.8                                                                        Positive for Enter-                 albicans                       bacter cloacae,                                                                            obacter cloacae and                                                1.5 × 10.sup.5                                                                       Candida albicans                                                   da albicans                             77 4    Light Klebsiella pneumoniae (2)                                                                   Normal Flora                                                                               >1 × 10.sup.8                                                                        Positive for Haemo-                                                philus, 1 × 10.sup.6                                                                  philus, possible                                                  Klebsiella pneu-                                                                           positive for Kleb-                                                 moniae and  siella pneumoniae                                                  Hormodendrum                                                                               and Pasteurella                                                    species, 1 × 10.sup.6                                                    Pasteurella                             78 3    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                79 2    Normal Flora       Normal Flora                                                                               Normal Flora                                                                               Normal Flora                80 3    Alpha streptococcus, Neisseria                                                                    Normal Flora                                                                               1 × 10.sup.8                                                                         Positive for Haemo-                                                philus, 1 × 10.sup.7                                                                  philus and Kleb-                                                  Klebsiella  siella pneumoniae                                                  pneumoniae                              __________________________________________________________________________

As can be seen in Table 2, the routine clinical method and the quantitative method (which utilized the saponin digestion step) were carried out on 80 sputum specimens, and 29 results differed. In 26 cases, the quantitative method found a positive when the routine clinical lab method did not, and in 3 cases, the routine clinical lab method found a positive whose numbers were not high enough to be considered a positive by quantitative standards. It is particularly noted that in 7 patients Haemophilus influenzae was found in the quantitative method on chocolate plates greater than 10⁷ per milliliter and was not detected by the routine clinical lab because of overgrowth. This particular organism has great clinical significance in pneumonia. Also, on four occasions, other organisms which constituted double infections were overgrown and not detected by the routine clinical laboratory analysis. Most of the differences in the results set forth in the table were in yeasts shown as positive by the quantitative method, and either not detected or considered light grown by the routine clinical laboratory method.

Next, to correlate laboratory findings with patient history, Table 3 shows a few correlations from the throat and lung autopsies. The results of these correlations is set forth in Table 3 below:

                  Table 3                                                          ______________________________________                                         Patient                                                                        No.   Culture     Results                                                      ______________________________________                                         I     Throat      Light Candida albicans                                             Sputum*     1 × 10.sup.5 Candida albicans, 1                                         × 10.sup.5                                                               Proteus and Pseudomomas aeruginosa                           II    Throat      Normal Flora                                                       Sputum      10.sup.8 Haemophilus, 10.sup.6 Klebsiella pneu-                                moniae, 10.sup.6 Pasturella                                  III   Sputum      1 × 10.sup.9 Klebsiella pneumoniae                           Throat      Moderate Candida albicans                                    IV    Throat      Light Pseudomonas aeruginosa                                       Sputum      1 × 10.sup.5 Pseudomonas aeruginosa                    V     Throat      Staphylococcus, Streptococcus Beta,                                            light Klebsiella pneumoniae                                        Sputum      10.sup.5 Klebsiella pneumoniae                                     Lung autopsy                                                                               Heavy Klebsiella pneumoniae                                  VI    Throat      Light Candida albicans                                             Sputum      >10.sup.6 Candida albicans, 10.sup.5 Serratia                                  Liquefaciens, 10.sup.5 Pseudomonas                           VII   Throat      Enterococcus, Escherichia coli                                     Sputum      Normal Flora                                                 VIII  Sputum      Normal Flora                                                       Lung autopsy                                                                               Enterbacter aerogenes                                        IX    Mouth       Heavy Klebsiella pneumoniae and                                                Candida tropicalis                                                 Throat      Heavy Klebsiella pneumoniae and                                                Candida tropicalis                                                 Tongue      Heavy Klebsiella pneumoniae and                                                Candida tropicalis                                                 Sputum      10.sup.8 Klebsiella pneumoniae, 10.sup.7 Entero-                               coccus                                                       X     Throat      Lactobacillus and Candida albicans                                 Sputum      10.sup.8 Klebsiella pneumoniae, 10.sup.8 Entero-                               bacter cloacae, 10.sup.5 Candida kruseii, 10.sup.4                             Candida tropicalis, 10.sup.5 Torulopsis                                        glabrata                                                     XI    Throat      Moderate Staphylococcus aureus                                     Sputum      1 × 10.sup.7 Staphylococcus aureus                     XII   Throat      Heavy Klebsiella pneumoniae and Entero-                                        bacter aerogenes, Candida albicans                                 Sputum      10.sup.8 Enterobacter aerogenes and 10.sup.5                                   Candida albicans                                                   Blood autopsy                                                                              Enterobacter aerogenes and Pseudomonas                       XIII  Throat      Normal Flora                                                       Sputum      10.sup.4 Candida tropicalis, 10.sup.4 Torulopsis                               glabrata                                                     ______________________________________                                          *quantitative sputum analysis                                            

As can be seen from Table 3, in some cases the throat has the same organisms at the sputum which means that the patient was either colonized or there was perhaps oral contamination in the sputum sample. Repeat cultures and observations on changing counts would possibly enable the physician to differentiate between these two latter possibilities. If colonization is observed, precautions could then be taken to prevent the establishment of a lung infection. Furthermore, in many instances, the quantitative sputum analysis reveals heavy infection wherein the throat analysis of the throat specimen does not. In this latter case, the data would support the conclusion that there is minimal nasopharyngeal contamination and that a lung infection in this case is highly probable.

EXAMPLE III

This Example is presented to show the mucolytic action (ability to degrade viscosity) of saponin on a clinical sputum specimen. The sputum specimen was 2 milliliters of a grade 2 sputum as defined in Example II above. To measure the viscosity of the sample, a small viscometer was built such as described in "A Simple Method of Measuring Sputum Viscosity", A. O. Jenssen, Scand. J. Resp. Dis.; 54, 290-296 (1973). This viscometer comprises a single plastic block, the bottom face of which was ground plane. In this bottom face, a canal, semicircular in section, was drilled out, ending in a vertical bore adapted to top to a Y fitting, one branch being connected via plastic hose to an air flow regulator, and the other branch of the Y fitting was connected to a water nonometer via plastic tube. An air escape port was drilled into the vertical bore just above the canal. Changes in pressure were measured as a decrease in the height of the water column in the nonometer. The time required to void the viscometer was recorded in seconds. To measure the viscosity of a given sample, the sputum was placed on a glass plate. The viscometer, with constant air flow, was placed on the sputum filling the channel of the viscometer. An air escape port on the side of the viscometer was closed. The time required to empty the viscometer and the maximum deflection of the height of the water column were recorded. The change in height in milliliters of the water column was converted to millilmeters of mercury (pressure) by multiplication by 0.00881. Next, according to the Jenssen article, pressure x seconds x K = viscosity, where K is a calibration constant for the viscometer. The viscometer was calibrated against an Oswald viscometer using glycerol as a primary standard. For this viscometer, K = 13.5 × 10³. The viscosities obtained with grade 2 sputum with and without addition of the purified saponin described in Example I and II are shown in Table 4 below:

                  Table 4                                                          ______________________________________                                         Mucolytic Action of Saponin on a                                               Clinical Sputum Specimen                                                       Nature of sputum: 2 ml of a grade 2.0 sputum.                                                      Centipoise                                                 ______________________________________                                         A.    Viscosity of 5 different                                                                              881                                                     portions before addition of                                                                         1,332                                                     saponin:            14,295                                                                         19,929                                                                          6,962                                                                         X--  = 8678                                                                    s.d. = 8306                                          B.    Viscosity of 4 different                                                                             166                                                      portions after the addition                                                                          177                                                      of 0.1 ml of 13% saponin:                                                                            161                                                                            172                                                                          X--  = 169                                                                     s.d. = 7                                             ______________________________________                                    

As can be seen, the action of the saponin effectively digested the sputum and resulted in a substantially uniform viscosity of all portions.

While the invention has been described in relation to its preferred embodiments, it is to be understood that various modifications thereof will now be apparent to one of ordinary skill in the art upon reading this specification and it is intended to cover such modifications as fall within the scope of the appended claims. 

I claim:
 1. In a method of detecting the presence of pathogenic organisms in the respiratory tract wherein a sample of lung fluid is obtained and thereafter analyzed for the presence of microbial pathogens, the improvement comprising:admixing said sample of lung fluid with an effective mucolytic amount to degrade the viscosity of said lung fluid sample and to impart a substantially uniform viscosity to said sample of lung fluid of a nontoxic saponin which has had microbial toxins removed therefrom which exhibit an apparent molecular weight of less than about 600 in an aqueous solution and thereafter thoroughly admixing the resulting sample having said substantially uniform viscosity to uniformly disperse said microbial pathogens therewithin prior to the time that said sample is analyzed.
 2. The improved method of claim 1 wherein said nontoxic saponin is admixed with said sample of lung fluid in an amount ranging from about 0.1 to about 20% by weight of said mixtures of lung fluid and saponin.
 3. The improved method of claim 1 wherein said nontoxic saponin is admixed with said sample of lung fluid in an amount ranging from about 5 to about 10% by weight of said mixture of lung fluid and saponin.
 4. The improved method of claim 2 wherein said nontoxic saponin is contained within an aqueous solution.
 5. The improved method of claim 4 wherein said aqueous solution of nontoxic saponin further comprises a minor effective amount of an antifoaming agent which is nontoxic to microbial pathogens.
 6. The improved method of claim 5 wherein said sample is analyzed by plating portions of said samples after treatment with said saponin on growth media.
 7. In a method of detecting the presence of pathogenic organisms in the respiratory tract wherein a sputum sample is obtained and thereafter analyzed for the presence of microbial pathogens, the improvement comprising:admixing said sputum sample with an effective mucolytic amount to degrade the viscosity of said sputum sample of a nontoxic saponin which has had microbial toxins removed therefrom which exhibit an apparent molecular weight of less than about 600 in an aqueous solution and allowing the viscosity of said sputum sample to degrade prior to the time that said sample is analyzed.
 8. The improved method of claim 7 wherein said nontoxic saponin is admixed with said sputum sample in an amount ranging from about 0.1 to about 20% by weight of said mixture of sputum and saponin.
 9. The improved method of claim 1 wherein said nontoxic saponin is admixed with said sputum sample in an amount ranging from about 5 to about 10% by weight of said mixture of sputum and saponin.
 10. The improved method of claim 7 wherein said nontoxic saponin is contained within an aqueous solution.
 11. The improved method of claim 10 wherein said aqueous solution of nontoxic saponin further comprises a minor effective amount of an antifoaming agent which is nontoxic to microbial pathogens.
 12. The improved method of claim 11 wherein said sample is analyzed by plating portions of said samples after treatment with said saponin on growth media. 